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Mutant trehalose synthase as well as expression gene and application thereof

A technology of trehalose synthase and gene expression, which is applied in the field of genetic engineering, can solve the problems of difficulty in separation and purification of downstream products, increase the production cost of trehalose, etc., and achieve the effects of large output, good thermal stability and reduced separation cost

Active Publication Date: 2016-04-27
SHANGHAI CHANGING BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The catalytic efficiency and enzymatic properties of trehalose synthase from different microbial sources are different, but they are all accompanied by the generation of by-products during the reaction process, some as high as 20%, generally about 5%-10%, which is very important The separation and purification of downstream products brings difficulties and increases the production cost of trehalose

Method used

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  • Mutant trehalose synthase as well as expression gene and application thereof
  • Mutant trehalose synthase as well as expression gene and application thereof
  • Mutant trehalose synthase as well as expression gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: Cloning the mutated trehalose synthase gene.

[0033] The primers at the mutation point were designed according to the full-length nucleotide sequence of Pseudomonas stutzeri (Pseudomonasstutzeri) trehalose synthase published on NCBI, and the primer sequences were as follows:

[0034] Upstream primer: GTGGCCCTCCGACCAttcGGTGCC

[0035] Downstream primer: CGTGCCGAGGGCACCgaaTGGTCG

[0036] Using the constructed tres-pGLO1 plasmid as a template, use the above primers for PCR amplification. The PCR reaction system is as follows:

[0037]

[0038] The above-mentioned PCR reaction is carried out according to the following procedures:

[0039] Pre-denaturation at 98°C for 3min; denaturation at 98°C for 10s, annealing at 58°C for 30s, extension at 72°C for 4min, 25 cycles; final extension at 72°C for 10min.

[0040] After PCR, the fragment length was analyzed by 1wt% agarose gel electrophoresis.

Embodiment 2

[0041] Example 2: Transforming the mutated trehalose synthase gene into an expression host to obtain a positive expression strain.

[0042] The PCR product was cleaved by DpnI endonuclease, and the enzyme cleavage reaction system was as follows:

[0043] Enzyme digestion system for PCR products:

[0044]

[0045] Mix well and centrifuge for a few seconds, collect the droplet on the tube wall to the bottom of the tube, and react at 37°C for 30 minutes.

[0046] Transformation of recombinant plasmids

[0047] (1) Preparation of Competent Cells

[0048]①Pick a single colony of Escherichia coli BL21 (DE3) (purchased from Shanghai Qianchen Biotechnology Co., Ltd.) (or pick a preserved strain) and inoculate it into 10ml of liquid LB medium, culture overnight at 37°C and 210rpm;

[0049] ② Inoculate 5ml of bacterial liquid into 500ml of LB medium, shake at 37°C, 210rpm for 70-80min to OD 600 reached 0.375;

[0050] ③ Place the bacterial solution on the ice-water mixture for 1...

Embodiment 3

[0074] Example 3: Fermentation and culture of positive expression strains, isolation and purification of mutated trehalose synthase recombinant protein

[0075] Seed culture: Pick the positive clones prepared in Example 2 in a conventional way, place them in 5 mL of LB liquid medium containing 100 μg / L ampicillin, and culture them with shaking at 37°C for 5-6 hours;

[0076] Bacterial cell expansion culture: insert the seeds into 1L liquid medium containing 100mg ampicillin, shake and cultivate at 37°C until the bacterial concentration is OD 600 When the temperature is 1.0, lower the temperature to 16°C; add isopropylthiogalactopyranoside (IPTG) at a final concentration of 0.2mM after 1 hour, and induce expression overnight.

[0077] Collect the cells: 4200rpm, centrifuge at 4°C for 15min, discard the supernatant, and harvest the cells; add resuspension solution (25mM Tris-HCl, pH8.0, 200mMNaCl), shake and precipitate the cells; add protease inhibitor PMSF (Phenylmethylsulfony...

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Abstract

The invention relates to mutant trehalose synthase as well as an expression gene and application thereof. The nucleotide sequence of the expression gene of the mutant trehalose synthase is as shown in SEQ ID NO. 1. The amino acid sequence of the mutant trehalose synthase is as shown in SEQ ID NO. 2. On the basis of a three-dimensional structure predicted by pseudomonas stutzeri Qlu3, the invention conducts key amino acid site-specific mutagenesis on the active center for the first time, so that the mutant protein of the trehalose synthase is obtained; the mutant trehalose synthase is simple and convenient in preparation method, high in yield, high in purity and good in thermal stability; and compared with wild trehalose synthase, the trehalose conversion rate of the mutant is improved by 4.5% and the generation amount of glucose, as a byproduct, is reduced by 69.4%.

Description

technical field [0001] The invention relates to a mutant trehalose synthase and its expression gene and application, belonging to the technical field of genetic engineering. Background technique [0002] Trehalose is a very safe and reliable non-reducing disaccharide. Frederick took the lead in exploring its chemical structure by using nuclear magnetic resonance technology. There are two glucopyranose monomers linked by 1,1 glycosidic bonds. of disaccharides. In the first half of the nineteenth century, trehalose was first extracted from a rye ergot fungus by Victrex, and it has been confirmed that trehalose has non-specific protective effects on various biologically active substances. And because of its drought resistance, low temperature resistance, and severe cold resistance, it is called "sugar of life" by many people. Trehalose can enable organisms to endure harsh environmental conditions such as high temperature, high cold, high osmotic pressure, and dehydration due ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/61C12N9/90C12N15/70C12N1/21C12P19/24C12P19/12
CPCC12N9/90C12P19/12C12P19/24
Inventor 苏静李珍珍王瑞明张云霄
Owner SHANGHAI CHANGING BIOTECH CO LTD
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